Plant color is one of the most important characters from an industrial viewpoint. As seen from pursuit of diversified flower colors, good expression of fruit colors, stabilization and uniformity of expressed colors, and so on, plant color is a big economical factor in flowers and ornamental plant, fruit trees and vegetables. Among plant pigments, the most abundantly seen are compounds generically termed anthocyanin. Cells and tissues where anthocyanin is accumulated present various colors from light blue to dark red. To date, almost 500 types of anthocyanin have been reported from various plant species, and their colors are mainly depending on the chemical structures thereof. Anthocyanidin (an aglycone that is the skeleton of anthocyanin) does not exist in plant bodies as it is, but exists in a modified form which has undergone glucosylation or acylation. Through glucosylation, anthocyanidin becomes nontoxic anthocyanin and stabilized; also, it becomes water-soluble and dissolved in cell vacuoles. A large number of glycosylated anthocyanins undergo further modification such as glycosylation, acylation or methylation. In particular, acylation increases the stability of anthocyanin molecules in vacuoles. Acylation by an aromatic acyl group bathochromically shifts the UV/visible absorption maximum of anthocyanins as a result of intramolecular association of the aglycone and the aromatic acyl group(s). Therefore, plant tissues with accumulation of acylated anthocyanin with aromatic acyl groups present a purple to blue color in many occasions. Anthocyanins form complex pigments through intramolecular association with aromatic acyl groups; intermolecular association with co-pigments (such as acylglucose, flavone or flavonol) or metal ions; coordinate bond with metal ions; bond with polypeptides; etc. in vacuoles and presents diversified colors. Therefore, acylation of anthocyanin is one of the important chemical reactions in expanding the diversity of flower colors with anthocyanin.
Color expression with anthocyanin, a character which can be directly confirmed with eyes, has become a target for a great number of genetic, biochemical and molecular biological studies. To date, genes involved in flavonoid including anthocyanin biosynthesis have been cloned from floricultural plants and experimental model plants such as petunia (Petunia x hybrida), snapdragon (Antirrhinum majus), morning glory (Pharbitis nil or Ipomoea nil), Arabidopsis thaliana; fruits such as apple (Malus x domestica), grape (Vitis vinifera); vegetables such as egg plant (Solanum melongena), perilla (Perilla frutescens); and so on. The mechanism of flower color expression with anthocyanin is now being elucidated by analysis by means of natural product chemistry and physiology.
Difference in color expression via accumulation of anthocyanin in plant species such as gentian (Gentiana spp.), prairie gentian (Eustoma grandiflorum), morning glory, lobelia (Lobelia erinus), verbena (Verbena x hybrida) or cineraria (Senecio cruentus or Pericallis cruenta) is basically attributable to difference in the aglycone (pelargonidin, cyanidin, delphinidin, petunidin, malvidin, etc.) of anthocyanin, and it is known that accumulation of delphinidin-type pigments is effective for blue color expression. On the other hand, difference in flower color expression in plants such as petunia, delphinium (Delphinium spp.) or butterfly pea (Clitoria ternatea) is attributable to difference in the binding pattern of sugar and acyl group to anthocyanidin and the number of bonds thereof. Acyl groups do not directly bind to anthocyanin aglycone; in many cases they bind to sugar residues (such as glucose) bound to anthocyanidin. It is reported that the caffeoyl group (an aromatic acyl group) binding to a glucosyl group at position 3′ of anthocyanin B ring in Gentiana triflora and the p-coumaroyl group (an aromatic acyl group) binding to glucosyl groups at positions 3′ and 5′ in butterfly pea and Dianella spp. are intramolecularly associating with anthocyanin aglycone at closer positions than other aromatic acyl groups binding to other glucosyl groups at positions 3, 5, 7, etc. (Yoshida et al., (2000) Phytochemistry 54: 85-92; Terahara et al., (1996) Journal of Natural Products 59: 139-144; Bloor (2001) Phytochemistry 58: 923-927). Therefore, it is reasonably presumed that modification of glucosyl groups at positions 3′ and 5′ of anthocyanin with aromatic acyl groups will be able to express purple or blue colors in cells, tissues and organs of plants. However, genes to be used for such a purpose have not been isolated yet.
With respect to acylation of anthocyanin, there have been reported acylation with aliphatic acyl groups (such as acetyl, malyl, malonyl, methylmalonyl or succinyl) and acylation with aromatic acyl groups (such as p-coumaroyl, caffeoyl, feruloyl, sinapoyl, p-hydroxybenzoyl or galloyl).
As a gene encoding acylation of anthocyanin sugar residue with an aliphatic acyl group, there is reported a gene encoding a protein having an activity of transferring a malonyl group to a sugar residue at position 3 of flavonoid using an aliphatic acyl-CoA thioester as an acyldonor in dahlia (Dahlia variabilis) (Suzuki et al., (2002) Plant Physiology 13: 2142-2151; Japanese Unexamined Patent Publication No. 2002-233381), cineraria (PCT/WO96/25500; Suzuki et al., (2003) Plant Biotechnology 20: 229-234), chrysanthemum (Dendranthema x morifolium) (Suzuki et al., (2004) Plant Science 166: 89-96) and verbena and deadnettle (Lamium purpureum) (Suzuki et al., (2004) Journal of Molecular Catalysis B: Enzymatic 28: 87-93). Further, a gene encoding a protein having an activity of transferring a malonyl group to a sugar residue at position 5 of flavonoid using aliphatic acyl-CoA thioester as an acyl donor has been reported from Salvia splendens (Suzuki et al., (2001) Journal of Biological Chemistry 276: 49013-49019; Suzuki et al., (2004) Plant Journal 38: 994-1003; PCT/WO01/92536); and Salvia guaranitica, lavender (Lavendula angustifolia) and perilla (PCT/JP01/04677).
Further, as a gene encoding acylation of anthocyanin sugar residue with an aromatic acyl group, there is reported a gene encoding a protein having an activity of transferring an aromatic acyl group to a sugar residue at position 3 of flavonoid using aromatic acyl-CoA thioester as an acyl donor in perilla and lavender (PCT/WO96/125500; Yonekura-Sakakibara et al., (2000) Plant Cell Physiology 41: 495-502) and petunia (PCT/WO01/72984). Still further, a gene encoding a protein having an activity of transferring an aromatic acyl group to a sugar residue at position 5 of flavonoid using an aromatic acyl-CoA thioester as an acyl donor in Gentiana triflora (PCT/WO96/25500; Fujiwara et al., (1998) Plant Journal 16: 421-431) and prairie gentian (Noda et al., (2000) Breeding Research 3 (Supplement 1): 61; Noda et al., (2002) The 20th Annual Meeting of the Japanese Society of Plant Cell and Molecular Biology: Abstract: 145).
Thus, those reported genes and its proteins catalyzes the acyl transfer to the anthocyanin sugar residues using acyl-CoA thioester as acyl-donor. However, it is reported that acyl donors include, in addition to acyl-CoA thioester, chlorogenic acid and 1-O -acyl-β-D-glucose (Steffens (2000) Plant Cell 12: 1253-1255).
With respect to proteins having an acyl transfer activity using chlorogenic acid as an acyl donor, purification and biochemical analysis of chlorogenic acid:glucaric acid caffeoyltransferase (5-O-caffeoylquinic acid:glucaric acid caffeoyltransferase) from tomato (Lycopersicon esculentum) have been reported (Strack and Gross (1990) Plant Physiology 92: 41-47).
As proteins having an activity of 1-O-acyl-β-D-glucose dependent acyltransferase activity, the following reports have been made. With respect to choline sinapoyltransferase involved in 1-O-sinapic acid ester metabolism (1-O-sinapoyl-β-D-glucose:choline 1-O-sinapoyltransferase), partial purification and characterization from seeds of wild radish (Raphanus sativus) and white mustard (Sinapis alba) (Gräwe and Strack (1986) Zeitschrift für Naturforchung 43c: 28-33); analysis of Arabidopsis thaliana mutants and cloning of the gene (Shirley et al., (2001) Plant Journal 28:83-94) and biochemical analysis using a recombinant protein (Shirley and Chapple (2003) Journal of Biological Chemistry 278: 19870-19877); and cloning of SNG2 gene from Brassica napus (Milkowski et al., (2004) Plant Journal 38: 80-92) have been reported.
With respect to malate sinapoyltransferase involved in sinapic acid ester metabolism (1-O-sinapoyl-β-D-glucose:malate 1-O-sinapoyltransferase), localization in Raphanus sativus cells (Sharma and Strack (1985) Planta 163: 563-568), measurement of activity in Brassica napus seeds and seedlings (Strack et al., (1990) Planta 180: 217-219), measurement of enzyme activity in seedlings and plantlets of Arabidopsis thaliana and Brassica rapa ssp. oleifera (Mock et al., (1992) Zeitschrift für Naturforchung 47c: 680-682), protein purification and biochemical analysis from wild radish hypocotyls (Gräwe et al., (1992) Planta 187: 236-241), analysis of Arabidopsis thaliana mutants and cloning of SNG1 gene (Lehfeldt et al., (2000) Plant Cell 12: 1295-1306; PCT/WO02/04614), and localization in cells of leaf tissue in Arabidopsis thaliana (Hause et al., (2002) Planta 215: 26-32) have been reported.
With respect to glucose acyltransferase involved in fatty acid metabolism (1-O-butyryl-β-D-glucose: 1-O-butyryl-β-D-glucose 2-O-butyryltransferase), measurement of enzyme activity in Lycopercsicon pennellii (Ghangas and Steffens (1995) Archives of Biochemistry and Biophysics 316: 370-377; Ghangas (1999) Phytochemistry 52: 785-792), purification and determination of partial amino acid sequences (Li et al., (1999) Plant Physiology 121:453-460) and cloning of gene (Li and Steffens (2000) PNAS 97: 6902-6907; PCT/WO97/48811) have been reported.
With respect to 1-O-indole-3-acetyl-β-D-glucose:myo-inositol indole-3-acetyltransferase involved in indoleacetic acid metabolism, measurement of enzyme activity from corn (Zea mays) (Michalczuk and Bandurski (1980) Biochemical Biophysics Research Communication 93: 588-592), and purification and biochemical analysis of protein and analysis of partial amino acid sequence (Kowalczyk et al., (2003) Physiologia Plantarum 119:165-174) have been reported.
With respect to 1-O-hydroxycinnamoyl-β-D-glucose:bethanidine diglucoside O-hydroxycinnamoyltransferase involved in betalain biosynthesis, detection of activity from suspension culture cells of wild spinach (Chenopodium rubrum) or petals of Lampranthus sociorum (Bokern and Strack (1988) Planta 174:101-105; Bokern et al., (1991) Planta 184: 261-270), and purification of protein and analysis of biochemical properties thereof (Bokern et al., (1992) Botanica Acta 105: 146-151) have been reported.
With respect to β-glucogallin (1-O-galloyl-β-D-glucose) dependent galloyltransferase involved in gallotannin biosynthesis, purification of protein and analysis of biochemical properties thereof from Stag's horn sumach (Rhus typhina) leaves (Niemetz and Gross (2001) Phytochemistry 58: 657-661; Fröhlich et al., (2002) Planta 216: 168-172) and English oak (Quercus robur) leaves (Gross et al., (1986) Journal of Plant Physiology 126: 173-179) have been reported.
As described above, purification of proteins having an activity of catalyzing acyl transfer reaction using 1-O-acyl-β-D-glucose as an acyl donor; elucidation of the biochemical properties of such proteins; and cloning of genes encoding such proteins have already been reported. However, with respect to detection of the activity of 1-O-acyl-β-D-glucose dependent acyltransferase that transfers an acyl group to sugar residues of flavonoids (such as anthocyanin), there has been only one report about 1-O-sinapoyl-β-D-glucose:anthocyanidin triglucoside sinapoyltransferase in cultured cells of carrot (Daucus carota) (Glaessgen and Seitz (1992) Planta 186: 582-585). Purification of such a protein with activity or cloning of genes encoding the same has not been performed yet.    [Patent Document 1] Japanese Unexamined Patent Publication No. 2002-233381    [Patent Document 2] PCT/WO 01/92536    [Patent Document 3] PCT/WO 01/72984    [Patent Document 4] PCT/WO 02/04614    [Patent Document 5] PCT/WO 97/48811